1. Field of the Invention
The invention relates to the preparation of mature HRV2 proteinase 2A, the partial purification thereof and the preparation of substrates having an inhibitory effect.
2. Background Information
Numerous animal and plant viruses require an input of vitally-coded proteinases during their replication cycle. The Picorna viruses, a family of significant viruses pathogenic to humans and animals, are for example totally dependent on proteolytic processing. The proteolytic enzymes involved in the replication are highly substrate-specific and generally recognise a cleavage region--i.e. a structurally determined recognition feature--and, to a lesser extent, an accurately defined amino acid pair such as is generally used as a recognition sequence. A general survey on this subject is provided by Krausslich, H. G. and Wimmer, E. (Ann. Rev. Biochem. 57:701-751 (1988)) and Kay, J. and Dunn, B. M. (Biochim. Biophys. Acta 1048:1-18 (1990)).
In view of their substrate specificity and their catalytic mechanism the viral proteinases constitute a good therapeutic point of attack (Johnston, M. I. et al., Trends Pharmacol. Sci. 10:305-307 (1989)). By preparing the three-dimensional structure of the proteinases of Rous Sarcoma Virus (Leis, J. et al., ASM-News 56:77-81 1990)) and of HIV I (Navia, M. A. et al., Nature 337:615-620 (1989); Miller, M. et al., Science 246:1149-1152 (1989)) it is possible to carry out computer-aided molecular designing of highly specific inhibitors (Meek, T. D. et al., Nature 343:90-92 (1990)). Specific proteinase inhibitors could therefore be new antiviral substances directed, for example, against viruses for which it is not possible to develop a vaccine for purely technical reasons; e.g., in the ease of rhinoviruses, there are at present well over 115 serotypes which do not crossreact with one another, 90 of which have already been classified as definite serotypes (Cooney, M. K. et al., Infect. Immun. 37:642-647 (1982)).
Proteolytic processing of the polyprotein (Jacobson, M. F. and Baltimore, D., Proc. Natl. Acad. Sci. 83:5392 (1968)) by vitally coded proteinases is preferably found in (+)-strain RNA viruses (Hellen, C. U. T. et al., Biochemistry 28:9881-9890 (1989)) and in retroviruses (Skalka, A. M., Cell 56:911-913 (1989)). By means of molecular biology studies, sequence comparisons and X-my structural analysis it has been found that the proteinases coded by viruses can be put into two categories of proteolytic enzymes, namely the pepsin-like aspartate proteinases (Meek, T. D. et al., Proc. Natl. Acad. Sci. 86:1841-1845 (1989)) and the cysteine-proteinases with trypsin-like protein chain folding (Bazan, J. F. and Fletterick, R. J., Proc. Natl. Acad. Sci. 85:7872-7876 (1988)). It is not always only virally coded proteinases which are involved in the proteolytic processing of viral polyprotein; for example, cellular proteinases participate in the maturation cleavage of the polyprotein of Yellow Fever Virus which belongs to Flaviviridae (Ruiz-Linares, A. et al., J. Virol. 63:4199-4209 (1989)).
The substrate specificity of the viral enzymes has been analysed more closely by detailed studies such as point mutation analysis, cleaving of peptide substrates in vitro and amino acid sequencing of the native cleavage products. It was found that, as already mentioned hereinbefore, it is less the cleavage site itself than some positions upstream or downstream which play a significant part in the recognition of cleavage sites. However, this sequence heterogeneity of the cleavage signals or their immediate environment results in a kind of "hierarchy" of the cleavage events in the polyprotein (Krausslich, H. G. et al., Proc. Natl. Acad. Sci. 86:807-811 (1989); Pichuantes, S. et al., PROTEINS: Structure, Function and Genetics 6:324-337 (1989); Darke, P. L. et al., J. Biol. Chem. 264:2307-2312 (1989); Nicklin, M. J. H. et al., J. Virol. 62:4586-4593 (1988); Libby, R. T. et al., Biochemistry 27:6262-6268 (1988); Sommergruber, W. et al., Virology 169:68-77 (1989)). The variation of the individual cleavage regions in the polyprotein thus permits a precisely determined sequence from kinetically "favorable" to kinetically "unfavorable" cleavings which then make it possible to carry out differential proteolysis of the individual cleavage products. Consequently, the viral proteinases have a kind of regulatory potential during the viral replication cycle. The principle of recognition of a specific secondary structure in the cleavage site area is not limited to Picorna viruses but would appear to be a general principle of viral proteinases. Thus, for example, these properties are also found in the adenovirus system (Webster, A. et al., J. Gen. Virol. 70:3225-3234 (1989); Webster, A. et al., J. Gen. Virol. 70:3215-3223 (1989) and in plant viral systems as well (Carrington, J. C. and Dougherty, W. G., Proc. Natl. Acad. Sci. 85:3391-3395 (1988); Dougherty, W. G. et al., EMBO J. 7:1281-1288 (1988)).
Hardly any other viral system is so dependent, for the regulation of the course of infection, on a controlled, limited proteolysis as the Picorna viridae system. This family of viruses can be subdivided into four different genera: entero-, rhino-, aphto- and cardioviruses. Rhinoviruses, like all other viruses of this family, are single-stranded (+)RNA viruses (Cooper, P. D. et al., Intervirology 10:165-180 (1978); MacNaughton, M. R., Current Top. Microbiol. Immunol. 97:1-26 (1982)). They are widespread, attack the upper respiratory tract in humans and cause acute infections which lead to catarrh, coughs, sore throat etc. and are generally referred to as colds (Stott, E. J. and Killington, R. A., Ann. Rev. Microbiol. 26:503-524 (1972)). Rhinovirus infections are among the commonest illnesses in man. The disease usually runs its course without any problems but, as the result of temporary weakening of the organism, there may be secondary infections caused by other viruses or bacteria which may in certain circumstances result in serious illness. Of the total of about 115 known different serotypes of human rhinoviruses, hitherto five serotypes (HRV 1B, 2, 9, 14 and 89) have been cloned and completely sequenced: German Patent Application P 35 05 148.5; Skern; T. et al., Nucleic Acids Res. 13:2111-2126 (1985); Duchler, M. et al., Proc. Natl. Acad. Sci. USA 84:2605-2609 (1987); Stanway, G. et al., Nucleic Acids Res. 12:7859-7877 (1984); Callahan, P. L. et al., Proc. Natl. Acad. Sci. USA 82:732-736 (1985); Hughes, R. et al., J. Gen. Virol. 69:49-58 (1988); Lecki, G. W., Ph.D. Thesis, University of Reading (1988)).
The genomie single-strand (+)RNA of the rhinoviruses is modified shortly after the infection by cleaving of the oligopeptide VPg bound to the 5' end and is subsequently used as mRNA for synthesizing a polyprotein which includes the entire continuous reading frame of the nucleic acid sequence (Butterworth, B. E., Virology 56:439-453 (1973); McLean, C. and Rueckert, R. R., J. Virol. 11:341-344 (1973); McLean, C. et al., J. Virol. 19:903-914 (1976); Agol, V. I., Prog. Med. Virol. 26:119-157 (1980); Putnak, J. R. and Phillips, B. A., Microbiol. Rev. 45:287-315 ( 1981 )). The mature viral proteins are formed solely by proteolytic cleaving from this polyprotein whilst--in the case of entero- and rhinoviruses at least--the proteinases which are effective are themselves part of this polyprotein. Processing is carried out in 3 stages (Palmenberg, A., J. Cell. Blochem. 33:191-198 (1987); Krausslich, H. G. and Wimmer, E., loc. cit. (1988)):
1.) primary cleavage: separation of the capsid precursor from the growing polypeptide chain;
2.) secondary cleavage: processing of structural and non-structural precursor proteins and
3.) mature cleavage of the capsid.
The first step therefore serves to cleave the precursor of the coat proteins and (in the case of entero- and rhinoviruses) is carried out autocatalytically by the proteinase 2A (cis-activity). The sequence of proteinase 2A (hereinafter referred to as 2A) is immediately after the section which codes for the coat proteins. Thus, in view of its location in the polyprotein, 2A is the first detectable enzymatic function of the virus. The separation of the coat protein region from the section responsible for replication occurs during the actual translation of the polyprotein "in statu nascendi". Experiments in vitro have shown that, in the poliovirus, this primary cleaving may be carried out at the P1-P2 region intermolecularly, i.e. "in trans" by the mature proteinase 2A (Krausslich, H. G. and Wimmer, E., loc. cit. (1988)). The cleavage signal which is thus recognized by the proteinase 2A has been, on the one hand, determined by direct amino acid sequence analysis of the N-terminus of 2A and/or the C-terminus of VP1 or, on the other hand, derived by comparison of the primary structure on the basis of homology studies. In polio (Pallansch, M. A. et al., J. Virol. 49:873-880 (1984)), BEV and HRV14 (Callahan, P. L. et al., loc. cit. (1985)) it is a Tyr/Gly amino acid pair, in HRV2 (Kowalski, H. et al., J. Gen. Virol. 86:3197-3200 (1987); Sommergruber, W. et al., Virology 169:68-77 (1989)) it is an Ala/Gly amino acid pair, in HRV1B (Hughes, P. J. et al., J. Gen. Virol. 69:49-58 (1988)) and HRV89 (Duchler, M. et al., loc. cit. (1987)) it is a Val/Gly pair and in Cox B1 (Iizuka, N. et al., Virology 156:64-73 (1987)), Cox B3 (Lindberg, A. M. et al., Virology 156:50-63 (1987)) and Cox B4 (Jenkins, O. et al., J. Gen. Virol. 68:1835-1848 (1987)) it is a Thr/Gly amino acid pair. This step is essential for the further progress of the viral infection (compartmentralization of replication and virus assembly). In cardioviruses and aphtoviruses, by contrast with the polioviruses, this cleaving is catalysed by proteinase 3C (Krausslich, H. -G. and Wimmer, E., loc. cit. (1988)). As regards the poliovirus system it is known that probably all the enzymes participating in this maturation cleavage are virally coded (Toyoda, H. et al., Cell 45:761-770 (1986)). In the poliovirus there are three types of cleavage signals; specific amino acid pairs Q-G which are recognized by the viral proteinase 3C (hereinafter referred to as 3C), the above-mentioned Y-G pair which serves as the 2A recognition signal and the N-S cleavage signal used in the mature cleavage of the capsid.
As has already been explained, the course of the infection in the case of picornaviruses is critically dependent on the viral enzymes. Since these very enzymes are particularly well conserved and are very similar in their properties in various rhinoviruses, they constitute an ideal target for chemotherapeutic intervention, e.g. the viral enzyme 2A. The chemotherapeutic point of attack is the inhibition of enzymatic activity by specific inhibitors. If the first proteolytic activity, the 2A activity, is inhibited, this prevents any further maturation of the viral system. Surprisingly, the 2A region of HRV2 has a marked homology not only with other rhinoviruses but also with viruses from other groups of the picornaviridae. An inhibitor against HRV2 2A might therefore be capable of being used on other picornaviruses.
The general importance of inhibiting virally coded proteinases has been moved back into the spotlight of possible antiviral therapeutic approaches not least by studies with the proteinase of human immunodeficiency virus 1 (HIV I). By deletion and point mutations in the proteinase region of this kind of retrovirus, it has been possible to recognise the essential role of the proteinase in the maturation of this type of virus (Katoh, I. et al., Virol. 145:280-292 (1985); Kohl, N. E. et al., Proc. Natl. Acad. Sci. USA 85:4686-4690 (1988); Crowford, S. and Goff, S. P., J. Virol. 53:899-907 (1985)). It has also been shown, by X-ray structural analysis and molecular biological studies, that the proteinase of HIV I belongs to the Asp-type, can process itself on the precursor protein (in recombinant prokaryotic systems as well), is capable of cleaving "in trans" specific peptides and occurs as an active proteinase in a homodimeric form (Navia, M. A. et al., loc. cit. (1989); Meek, T. D. et al., loc. cit. (1989); Katoh, I. et al., loc. cit. (1985)). In view of the fact that the proteinase of HIV I occurs as a dimer in its active form, Wlodawer and colleagues also proposed the development of specific dimerization inhibitors (Wlodawer, A. et al., Science 245:616-621 (1989)). The development of highly specific competitive inhibitors against the proteinase of HIV I on the basis of modified peptide substrates was described only recently by Tomasselli and colleagues (Tomasselli, A. G. et al., Biochem. 29:264-269 (1990)). It had been known for even longer that a fungicidal antibiotic, cerulenin, has an antiretroviral activity against Rous Sarcoma Virus and Murine Leukemia Virus (Goldfine, H. et al., Biochem. Biophys. Acad. 512:229-240 (1978); Katoh, I. et al., Virus Res. 5:265-276 (1986)). In the case of HIV I, it was possible to make a connection between the inhibitory effect of cerulenin and the inhibition in the proteolytic processing of the polyprotein of HIV I (Pal, R. et al., Proc. Natl. Acad. Sci. 85:9283-9286 (1988)). Starting from this fact, Blumenstein and colleagues were able to develop specific inhibitors against proteinase HIV I on the basis of synthetic non-peptide inhibitors. In other words, they were able to trace the inhibitory effect of cerulenin to the interaction of the electrophilic epoxide group with nucleophilic regions of the proteinase. Moreover, as a result of the development of synthetic derivatives, the original toxicity of cerulenin has been reduced (Blumenstein, J. J. et al., Biochem. Biophys. Res. Commun. 163:980-987 (1989)).
Also in the picornaviral system, all kinds of organic or inorganic compounds as well as peptide derivatives and proteins are now known which have an inhibitory effect on the proteolytie processing of these viruses. The effect of these substances is based on the direct interaction with the proteinases (Kettner, C. A. et al., U.S. Pat. No.: 4,652,552 (1987); Korant, B. D. et al., J. Cell. Blochem. 32:91-95 (1986) and/or on the indirect route of interaction with substrates of these proteinases (Geist, F. C. et al., Antimicrob. Agents Chemother. 31:622-624 (1987); Perrin, D. D. and Stunzl, H., Viral Chemotherapy 1:288-189 (1984)). The problem with the majority of these substances is the relatively high concentration required for inhibition and the toxicity of these compounds, which is considerable with some of them. The already successful use of modified peptides and peptidomimeties as therapeutic agents in non-viral areas (Fauchere, J. L., Advanc. Drug Res. 15:29-69 (1986)) and inhibitor designing starting from known structures (DesJarlais, R. L. et al., "Viral Proteinases as Targets for Chemotherapy", in Curr. Commun. Mol. Biol., Cold Spring Harbor Laboratory Press, 203-210 (1989)) and the increase in molecular biological and physical data on picornaviral proteases has increased the understanding of the structure and function of these viral enzymes and thus permits a stepwise rational designing--starting from modified peptide substrates--to achieve highly specific and non-toxic peptidomimetics.
The first evidence of a second virally coded proteinase, 2A, came from antibodies developed against polio P3C which clearly suppressed all the cleaving carried out at Q-G but did not suppress cleaving between Y-G (Hanecak, R. et al., Proc. Natl. Acad. Sci. USA 79:3973-3977 (1982)). This observation lead to the conclusion that the proteolytic processing at Y-G sites requires its own proteinase. The seat of this second proteolytic activity could clearly be ascribed to 2A in poliovirus. It was interesting to discover 2A carries out alternative cleaving in the proteinase-polymerase region (3CD), which also occurs at a Y-G site and yields inactive enzymes 3C' and 3D'. This cleaving possibly serves to regulate the quantities of the enzymes 3C and 3D (Lee, C. K. and Wireruer, E., Virol. 166:1435-1441 (1988)). Since the synthesis of host protein is very rapidly stopped during infection with poliovirus in HeLa cells, but the translation of the poliovirus RNA is able to proceed unhindered, it was assumed that one or more regulating factors of the translation were changed during the infection. In fact, earlier findings show that the eukaryotic initiation factor 4F is changed by the proteolytic cleaving of the p220 component during the poliovirus infection in HeLa cells (Etchison, D. et al., J. Virol. 51:832-837 (1984); Etchison, D. et at., J. Biol. Chem. 257:14806-14810 (1982), Etchison, D. and Etchison, J. R., J. Virol. 61:2702-2710 (1987)). It was subsequently demonstrated that 2A is indirectly responsible for this modification of p220 in infected cells (Krausslich, H. G. et al., J. Virol. 61:2711-2718 (1987); Lloyd, R. E. et al., Virol. 150:299-303 (1986); Lloyd, R. E. et al., J. Virol. 61:2450-2488 (1987)). The question of the "trans" activity of the proteinase 2A was first answered in the affirmative in the poliovirus system, by making insertions and deletions in 2A to express an unprocessed P1-P2 region in E. coli which was able to be cleaved from poliovirus, which either came from cells infected with poliovirus (Nicklin, M. J. H., et al., Proc. Natl. Acad. Sci. USA, 84:4002-4006 (1987)), or was translated "in vitro" (Krausslich, .-G. et al., J. Virol. 61:2711-2718 (1987)) or expressed in E. coli, or purified from cells infected with poliovirus (Konig, and Rosenwirth, B., J. Virol. 62:1243-1250 (1988)). Bernstein and colleagues were able to distinguish between a "cis"- and a "trans"- active form of the proteinase 2A using Poliovirus mutants. These routants contain an additional Leu between the 102 and 103 amino acids of 2A. This Leu insertion lead to a poorly replicating but still viable poliovirus. No cleavage of the cellular p220 molecules could be observed in HeLa cells infected with these mutants (Bernstein, H. D., et al., Mol. Cell. Biol. 5:2913-2923 (1985); Bernstein, H. D., et al., J. Virol. 60:1040-1049 (1986)).
With the aid of recombinant vaceinia vectors which contained the complete P1 region and the gene section for a shortened inactive form of proteinase 2A from polio, it was shown that after coinfection with one of the three serotypes of polio, with HRV14 or with EMCV both in the cases of polio 1, 2 and 3 and also in the case of HRV14, correct intermolecular ("trans") processing occurred at the P1/2A cutting site. After coinfection with EMCV, there was no processing of the inactive precursor protein of polio (Jewell, J. E., et al., J. Virol. 64:1388-1393 (1990)). Moreover, in higher eukaryotic cells it was demonstrated, by means of expression vectors under the control of the LTR region of HIV I and activated by means of the "tat" gene product, that 2A from poliovirus is the inducing agent in the proteolysis of p220 (Sun, X. H. and Baltimore, D., Proc. Natl. Acad. Sci. 86:2143-2146 (1989)). Krausslich and his collaborators also showed that although the presence of an active 2A is absolutely necessary for the proteolytic degradation of p220 during the host cell shutoff, this degradation is not directly carried out by 2A (Krausslich, H. G. et al., loc. cit. (1987)). It is currently presumed that the proteinase 2A is capable of activating a cellular enzyme which consequently brings about the cleaving of p220. Recent investigations have given rise to speculation that possibly the Ca.sup.2 + -dependent cellular proteinase calpain may be responsible for this "p220-ase" activity. According to this model, the proteinase 2A would convert an inactive calpain into an active form by cleaving it and the active form would then attack the p220. The possible cleaving of the calpain might occur 80 amino acids away from the N-terminus in the sequence Y-G (Wyckoff, E. E. and Ehrenfeld, E., EUROPIC 89, "Sixth Meeting of the European Study Group on the Molecular Biology of Picornaviruses," Bruges, Belgium (1989)).
Possibly, the two proteinases 2A and 3C are directly or indirectly involved in the proteolytic degradation of another 14 cellular proteins during the infection of HeLa cells with poliovirus (Urzainqui, A. and Carrasco, L., J. Virol. 63:4729-4735 (1989)).
The most recent data show that poliovirus proteinase 2A inhibits not only the translation of the host cell but also the DNA replication and the RNA polymerase II transcription (Davies, M. V., et al., J. Biol. Chem. 266:14714-14720 (1991)).
As a result of these data the various 2A proteinases of the individual entero- and rhinoviruses would have to recognise one or more common targets in the cell. Therefore, the understanding and knowledge of the trans-substrate specificity of 2A is of great importance.
Hitherto, it has only been possible to express the 2A of HRV2 as a fusion protein. As a result of the autocatalytic activity the mature proteinase is cleaved from the precursor molecule after expression. Owing to the poor solubility of the precursor protein and the cleaved proteinase itself, however, this process involves a significant loss of yield, which means that the mature proteinase released is present in a very small concentration (Sommergruber et al., loc. cit. (1989)).